Asbestos fibers damage lung mesothelial cells which triggers a compensatory proliferative response, inflammation, and malignant mesothelioma (MM). In this proposal we will focus on inflammasome components, NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and Caspase-1, which we show are attenuated in MM cells and tumors and activated by asbestos in mesothelial cells. Decreased levels of NLRP3 and caspase-1 may be responsible for enhanced drug resistance in these tumors. Here we hypothesize that constitutively attenuated levels of NLRP3 and caspase-1 activity in MM cells and tumor tissues impart enhanced drug resistance to this tumor. We also hypothesize that chronic exposure to asbestos leading to MM tumorigenesis results in downregulation of NLRP3 expression and activity. In Specific Aim 1: A) We will assess expression and activity of NLRP3, ASC in various human MM cells and primary tumors as compared to mesothelial cells and normal matching tissues. In addition, protein expression levels of NLRP3 will also be identified in MM tumor tissue arrays by immunofluorescence as compared to benign tissues. B) We will also study the effect of asbestos exposure on inflammasome expression and activation in immortalized (LP9) and a primary isolate of human mesothelial cells (HMC). Along with caspase-1 activity, levels of IL-1, IL-18 and IL-33 will be assessed as measures of inflammasome activation. C) To show that IL-1 generated as a result of activation of inflammasomes by long term exposure to asbestos causes compensatory proliferation and transformation of mesothelial cells, we will treat LP9 cells with IL-1 for different time points and assess proliferation, markers of mesothelial to fibroblast transition (MFT) and colony formation ability of these cells in soft agar. Also, the IL-1 receptor blocker Anakinra, will also be used to confirm the role of IL-1 in these processes. In Specific Aim 2: We will study the role of long term asbestos exposure on NLRP3 regulation, to understand the cause of observed low NLRP3 levels in MMs, for this, we will A) determine the role of extracellular signal regulated kinase (ERK1/2) in inflammasome expression, activity and functional regulation in mesothelial and MM cells by using small molecule inhibitor, small interfering/short hairpin si/sh RNA as well as overexpression approaches, and B) evaluate if the NLRP3 promoter is methylated in MM cells and tumors and study if long term asbestos exposure causes methylation of the NLRP3 promoter in LP9 cells. Lastly in Specific Aim 3, we will evaluate the role of NLRP3 in MM tumor development using mouse models. A) Human MM cells stably overexpressing NLRP3 or empty vector will be injected intraperitoneally (IP) into SCID mice. Two weeks after cell injection mice will receive either saline or chemotherapeutic drugs (Doxorubicin, Cisplatin) or drugs plus Anakinra for 4-6 weeks. Effects of inflammation on the process of tumor development with and without drug will be assessed by collecting peritoneal lavage fluid (PLF) and determining differential cell counts and cytokine profiling. B) We will also cross NLRP3-/- mice with SCID mice to get SCID-NLRP3-/- mice. Using these mice, we will be able to study the effects of host NLRP3 protein on human MM tumorigenesis. The goal of these studies will be to demonstrate the role of inflammasomes in MM development and may help in designing future therapeutic strategies for patients. PUBLIC HEALTH RELEVANCE: Apoptosis plays a major role in tumor growth and drug resistance. Here we hypothesize that constitutive inhibition of NLRP3 component of inflammasomes and caspase-1 activity in malignant mesothelioma (MM) cells and tumors is responsible in part for the drug resistance. This project will use human mesothelial and MM cells to determine whether inflammasome activation and IL-1 release after asbestos exposure is responsible for their transformation to malignant phenotype. Mechanisms of asbestos-induced tumorigenesis and the involvement of inflammasomes in these processes will also be explored using in vitro approaches and established SCID mouse models injected with human cells. Results of this study may help in developing new therapeutic strategies for MM.